There is currently no effective treatment to improve axon regeneration in humans. Thus, basic research in model organisms such as nematodes, flies, and mice is needed to provide a better undertanding of the biological mechanisms that regulate regeneration. Recent work has demonstrated that the conserved DLK-1 signaling pathway is a critical regulator of regeneration. In nematodes, flies, and mice, the DLK-1 pathway regulates regeneration by modulating gene expression in injured neurons. This proposal seeks to identify the genes that are regulated by DLK-1 signaling (Aim 1), and to determine which of these genes are important for determining the regenerative potential of the injured neuron (Aim 2). These findings will expand understanding of the DLK-1 pathway, and have the potential to identify novel mechanisms for axon regeneration. This proposal uses an innovative approach in the model organism C. elegans to identify transcriptional targets of DLK-1 signaling that function in regeneration. Studies of gene transcription typically identify large numbers of targets, but it is often difficult to analyze the function of more than a few selected candidates. This proposal uses four strategies to address this difficulty. First, by using novel genetic backgrounds, analysis is focused on the transcriptional effects of a single signaling pathway-the DLK-1 pathway. Second, a novel approach is used to purify cells for transcriptional profiling, enabling analysis to be directed to a single neuronal type, the GABA motor neurons. Third, by using a novel RNAi technique, genes are knocked down only in GABA neurons for functional analysis, avoiding confounding effects and enabling the study of even essential genes. Fourth, functional analysis is performed using single-neuron laser axotomy in GABA neurons. These experiments will provide a detailed analysis of how modulation of gene expression by the DLK-1 pathway affects axon regeneration. In addition, this study will serve as a blueprint for future investigations into the mechanisms that link nerve injury, cellular signaling, gene transcription, and axon regeneration.